A novel method for simultaneous analysis of specific platelet antibodies: SASPA

Abstract

Anti-platelet antibodies cause several thrombocytopenic disorders such as autoimmune or alloimmune thrombocytopenia, post-transfusion purpura, drug-induced thrombocytopenia and refractoriness to platelet transfusion by eliminating platelets from the circulation. These antibodies can target specific platelet glycoproteins (GP), human leucocyte antigen (HLA) class I antigens or phospholipids (Kurata et al, 1993;Alcorta et al, 1996;Macchi et al, 1996Macchi et al, , 1997Cuadrado et al, 1997;Panzer et al, 1997;Kiefel et al, 2001;Ranasinghe et al, 2001;Cines & Blanchette, 2002). The detection of these antibodies with high sensitivity and characterization of their specificity is important for diagnosis and specific therapy.The platelet immunofluorescence test (PIFT) has been widely used as a screening method for the detection of platelet antibodies (von dem Borne et al, 1978;Christopoulos et al, 1993;Porcelijn and von dem Borne, 1998;Hagenström et al, 2000). In order to differentiate the detected antibodies, the monoclonal antibody-specific immobilization of platelet antigen (MAIPA) assay has been mostly used as the reference assay (Kiefel et al, 1987). Several other antibody-detection systems, such as immunoblotting, solid-phase radioimmunoassay and modified antigen-capture enzyme-linked immunosorbent assay (MACE), have been reported (Tijhuis et al, 1991;Hou et al, 1995). However, the drawbacks of these tests SummaryGlycoprotein (GP)-specific platelet antibodies can cause allo-immune and auto-immune thrombocytopenia. The specific detection of relevant antibodies is a prerequisite for diagnosis and treatment. Here, we describe an improved method based on simultaneous detection of various plateletspecific immunoglobulin G (IgG) and IgM antibodies. Bead populations with distinct fluorescence intensities, coated with monoclonal antibodies specific for mouse heavy chain isotypes, were used for the simultaneous immobilization of platelet-GP [IIb/IIIa, Ib/IX, human leucocyte antigen (HLA) class I, or Ia/IIa, CD32, GPIV or CD109, Ib/IX, HLA class I]. In order to detect human IgG and IgM antibodies simultaneously, phycoerythrin-and fluorescein isotiocyanate-conjugated goat anti-human IgG and IgM were added. On this basis, the abundance of six distinct antibodies (three anti-GP, each with subclasses IgG and IgM) were simultaneously analysed without cross-reaction by flow cytometry. For evaluation, sera and platelets from 169 patients with platelet-binding and/or platelet-associated antibodies were investigated. The monoclonal antibody-specific immobilization of platelet antigen (MAIPA) assay was performed in parallel as reference test. The simultaneous analysis of platelet-specific antibodies (SASPA) assay was able to detect all platelet-specific IgG and IgM that were also recognized by MAIPA with a comparable sensitivity. SASPA proved to be a rapid and reliable assay that required less platelets than other methods. This method has the potential to pave the way for new investigations of platelet-specific antibodies.

The SASPA (Simultaneous Analysis of Specific Platelet Antibodies) Assay: Implementation and Performance in the Routine Laboratory Use

“…Moreover, the multiple antibody detection by SASPA reduced working time to perform the assay. In a comparative investigation of SASPA and MAIPA, both direct and indirect, we demonstrated the same specificity for both methods [15]. Half of the patients with platelet-associated IgG (PAIgG) determined by PIFT remained negative in the GP-specific assays focusing on GP Ia/IIa, Ib/IX, IIb/IIIa as performed to date by MAIPA [14,16].…”

“…In addition, sensitivity of the SASPA assay was at least similar or even higher compared to the MAIPA assay. Studies on the stability of SASPA results showed lower coefficients of variation compared to MAIPA results, indicating that SASPA is more robust [15].…”

“…So far, we did not observe any inhibitory interference caused by the simultaneous use of three mAbs [15]. The evaluations of IgG and IgM antibodies are given as percentages and mean fluorescence intensities (MFIs).…”

“…However, this test could not be introduced into routine use due to difficulties of standardization. In 2004 we introduced a flow cytometric assay for simultaneous detection and differentiation of platelet-specific IgG and IgM antibodies (SASPA) [15]. In this article, we summarize the benefits of the SASPA method compared to the MAIPA assay and describe the implementation of the SASPA assay in routine laboratory use.…”

New Bioanalytical Technologies in Platelet Research and Diagnostics

“…Significant progress has also been achieved in the diagnostic field of platelet immunology. The recent introduction of the simultaneous analysis of specific platelet antibodies (SASPA) assay [12] and of recombinant platelet antigens [13] represent the most important developments. Such methods may contribute to gain knowledge about the heterogeneity of plateletspecific antigens and antibodies which represents a significant problem in the clinical diagnosis of immunologically mediated platelet disorders.…”